Serine/Threonine Protein Phosphatase 5 (Trypanosoma brucei)
PDB: 1S95
*NCBI Gene # or RefSeq#:
3657550
*Protein ID (NP or XP #) or Wolbachia#:
Tb927.10.13670
*Organism:
T. brucei
Etiologic Risk Group (see link below):
Risk 2
*Background/Disease Information:
Trypanosoma brucei causes the parasitic disease African trypanomiasis. This disease can infect both humans and animals, and is characterized by fever and headaches, until the lymphatic and circulatory systems become affected. The disease then progresses rapidly and has a variety of manifestations including severely disrupted sleep cycles due to the parasite passing through the blood brain barrier, anemia, endocrine malfunctions, cardiac problems and kidney dysfunctions.
The disease begins in the midgut of the fly where it eventually makes its way to the salivary glands of the fly. The fly will then bite a mammalian host (Human, monkey, etc.) and spread the disease.
Druggable Target: A similar target has been used in successful runs by the study alsford, and the TDR gives it a druggability index of 0.5. It displays some similarity to a human, but not an exact match with 49% similarity on a 98% query coverage. http://www.tdrtargets.org/targets/view?gene_id=19618
reaction3.1.3.16(2).PNG
--Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or other company) page for assay (see Sigma links below)
-- -or link (or citation) to paper that contains assay information
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model:
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage: 99%
---- Max % Identities: 49%
---- % Positives 68
---- Chain used for homology:
BlastP.PNG
Current Inhibitors:
Only 5 hits were found in the binding database, the top hit being CHEMBL318778.
Expression Information: The protein is usually expressed via its natural protozoa, but has been cloned and characterized from e.coli before. http://www.brenda-enzymes.org/literature/lit.php4?e=3.1.3.16&r=655670
Expression Information (has it been expressed in bacterial cells):
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
*Target (protein/gene names):Serine/Threonine Protein Phosphatase 5 (Trypanosoma brucei)
PDB: 1S95
*NCBI Gene # or RefSeq#:
3657550
*Protein ID (NP or XP #) or Wolbachia#:
Tb927.10.13670
*Organism:
T. brucei
Etiologic Risk Group (see link below):
Risk 2
*Background/Disease Information:
Trypanosoma brucei causes the parasitic disease African trypanomiasis. This disease can infect both humans and animals, and is characterized by fever and headaches, until the lymphatic and circulatory systems become affected. The disease then progresses rapidly and has a variety of manifestations including severely disrupted sleep cycles due to the parasite passing through the blood brain barrier, anemia, endocrine malfunctions, cardiac problems and kidney dysfunctions.
The disease begins in the midgut of the fly where it eventually makes its way to the salivary glands of the fly. The fly will then bite a mammalian host (Human, monkey, etc.) and spread the disease.
Link to TDR Targets page (if present):
http://www.tdrtargets.org/targets/view?gene_id=19618
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
Essentiality of this protein:
*EC#:
3.1.3.16
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.16&Suchword=&organism[]=Trypanosoma+brucei&show_tm=0
Complex of proteins?:
Druggable Target: A similar target has been used in successful runs by the study alsford, and the TDR gives it a druggability index of 0.5. It displays some similarity to a human, but not an exact match with 49% similarity on a 98% query coverage.
http://www.tdrtargets.org/targets/view?gene_id=19618
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or other company) page for assay (see Sigma links below)
-- -or link (or citation) to paper that contains assay information
http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/learning-center/assay-library/ec-number-iii.html
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/calcineurin.pdf
-- links to assay reagents (substrates) pages.
--- List cost and quantity of substrate reagents, supplier, and catalog #
Alkaline Phosphatase Yellow (pNPP) Liquid Substrate System for ELISA: $74.20
http://www.sigmaaldrich.com/catalog/product/sigma/p7998?lang=en®ion=US
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model:
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage: 99%
---- Max % Identities: 49%
---- % Positives 68
---- Chain used for homology:
Current Inhibitors:
Only 5 hits were found in the binding database, the top hit being CHEMBL318778.
Expression Information: The protein is usually expressed via its natural protozoa, but has been cloned and characterized from e.coli before.
http://www.brenda-enzymes.org/literature/lit.php4?e=3.1.3.16&r=655670
Expression Information (has it been expressed in bacterial cells):
Purification Method:
microcystin-Sepharose bead chromatography
http://www.brenda-enzymes.org/literature/lit.php4?e=3.1.3.16&r=694537
Image of protein (PyMol with features delineated and shown separately):
Image of protein:
Cartoon image of 1WAO, the closest protein resembling the chosen target.
http://tritrypdb.org/tritrypdb/showRecord.do?name=GeneRecordClasses.GeneRecordClass&primary_key=Tb927.10.13670
http://www.rcsb.org/pdb/explore/explore.do?pdbId=1wao
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MAGVEEADKLKQLGNAAFSERKWHLAIDMYTKAIELTKTPTLFCNRALAELRAELPGAAL
ADADAALGIEPTFAKAYYHKASAYLSLGKHKQALTNYKKVVDLAPQNSDAQAKVEFCKKE
IRRINFENAIMTPDEAPLSQTIKLGSVRADYDGPRIENETVTVEFVEAMKEHFRLEKLID
RHDVIFILLEVQKILKKCPNFVSINVPVGEEITVCGDTHGQYYDLLNIFKLNGNPLETNR
YLFNGDFVDRGSYSFENIMTLFAYKVLYPDHFFLSRGNHEGVSMNRMYGFEGEVTQKYNS
EMFRLFTEVFNSLPIGHIINNEVFVVHGGLYSSDKVTLDDLQHPNRFRDIPESGLICESL
WSDPQPMPGRAPSKRGVSCLSFGPDVTETFLNNNNLKLLVRSHEVKDEGYEIEHGGKCIT
VFSAPNYCDQMGNKGAFIRFTGGDMKPRFTTFTHVPHPGKRPMHYATGFGLF
*length of your protein in Amino Acids
1 <span class="ff_line">magveeadkl kqlgnaafse rkwhlaidmy tkaieltktp tlfcnralae lraelpgaal</span> 61 <span class="ff_line">adadaalgie ptfakayyhk asaylslgkh kqaltnykkv vdlapqntda qakvefckke</span> 121 <span class="ff_line">irrinfenai ttpdeaplsq tiklgsvrad ydgprienet vtvelveamk ehfrleklid</span> 181 <span class="ff_line">rhdvifille vqkilkkcpn fvsinvpvge ditvcgdthg qyydllnifk lngnpletnr</span> 241 <span class="ff_line">ylfngdfvdr gsysfgnimt lfaykvlypd hfflsrgnqe gvsmnrmygf egevtqkyns</span> 301 <span class="ff_line">emfrlftevf nsppighinn nevfvlhggl yssdkvtldd lqqpnrfrdi pesglicesl</span> 361 <span class="ff_line">wsdpqpmpgr apskrgvscl sfgpdvtetf lnnnnlkllv rshevkdegy eiehggkcit</span> 421 <span class="ff_line">vfsapnycdq mgnkgafirf tggdmkprft tfthvphpgk rpmhyatgfg lf</span>472
http://blast.ncbi.nlm.nih.gov/Blast.cgi#146083777
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website
53312.7
Molar Extinction coefficient of your protein at 280 nm wavelength:
38320
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
1 <span class="ff_line">tattaaaaga acagtttctg tactatattg cattggctgg aaggtcgtgt tgcg</span><span class="feat_h">atggcc</span> 61 <span class="feat_h">ggggtagagg aagcagacaa gctgaagcag ctgggaaacg cagcctttag tgagcgcaag</span> 121 <span class="feat_h">tggcaccttg ccattgacat gtatacgaag gctatcgaac tgacaaaaac gccaacattg</span> 181 <span class="feat_h">ttttgcaacc gtgcacttgc agagcttaga gctgagcttc ccggtgcggc gcttgctgac</span> 241 <span class="feat_h">gctgatgcgg cgttagggat agagccaacc ttcgcaaagg cctattacca caaggcatcc</span> 301 <span class="feat_h">gcgtatctga gccttggaaa acacaagcag gcgctgacaa actacaagaa agttgtagat</span> 361 <span class="feat_h">ttggcaccac aaaataccga tgcgcaggcg aaggtggagt tttgtaagaa ggaaatacgc</span> 421 <span class="feat_h">agaatcaact ttgaaaatgc cattacgact ccagacgagg caccgctgtc gcagacaatt</span> 481 <span class="feat_h">aaactgggca gtgtgcgtgc tgattacgat gggcctcgta tcgaaaatga aaccgtcacc</span> 541 <span class="feat_h">gtagaactcg tggaagcgat gaaggagcat ttcaggttag agaaactcat tgaccggcac</span> 601 <span class="feat_h">gacgttatat ttattctgtt ggaggtgcag aagattctga aaaaatgccc caactttgtg</span> 661 <span class="feat_h">tccatcaacg tgccagttgg tgaagacata acagtatgtg gtgacactca cgggcagtat</span> 721 <span class="feat_h">tacgatctcc tcaatatatt caagttgaac gggaatccct tggaaacgaa ccggtacctt</span> 781 <span class="feat_h">ttcaacggtg actttgtgga ccgcggttcg tactcatttg gaaatatcat gactctgttt</span> 841 <span class="feat_h">gcgtataaag tactttaccc ggatcacttt ttcttatctc gtgggaacca agagggtgtt</span> 901 <span class="feat_h">tccatgaacc gaatgtatgg gtttgaaggg gaagtaaccc aaaagtacaa cagcgaaatg</span> 961 <span class="feat_h">tttcgcctct tcacggaggt attcaacagt cctccaatcg gtcacattaa taacaacgag</span> 1021 <span class="feat_h">gtgtttgtac tgcacggcgg tctttactcc tcggataagg tgacattgga tgacctgcaa</span> 1081 <span class="feat_h">caacctaacc gattccgtga tatacctgag agcggtctaa tctgcgaaag cctgtggtcc</span> 1141 <span class="feat_h">gatccacagc cgatgcctgg gcgagcccca agtaagcgtg gggtgagctg cctgtctttc</span> 1201 <span class="feat_h">gggccggacg taaccgagac gttcctaaac aataacaacc ttaaactgct cgtacgctct</span> 1261 <span class="feat_h">catgaggtaa aggacgaggg gtacgagata gaacacggtg gcaaatgcat tacagtcttc</span> 1321 <span class="feat_h">agcgcaccaa actattgtga ccaaatgggc aacaagggag cattcatccg ttttaccggc</span> 1381 <span class="feat_h">ggtgacatga aaccaagatt cacaacattc acgcacgtgc cgcatcctgg gaaacggcca</span> 1441 <span class="feat_h">atgcattacg ccacaggatt tggcctgttt tga</span><span class="ff_line">ggaagag taaaaaggaa tggagggaaa</span> 1501 <span class="ff_line">gcgagacatc gcaggggaga gctgtgacca aagccaatgc acccgtgaca aggggcggcg</span> 1561 <span class="ff_line">ggggaaatat cattgggaaa tatattagtt aggatgcgta tatgcatgtg tatttgtgta</span> 1621 <span class="ff_line">tatatgcatg tgtgtaagca tcgttgaaat atgccttctc ttcctgcgca ggtatcgttc</span> 1681 <span class="ff_line">cttcgttgga gcctctgttc ttcagacatc ttccgtgcgc aggaggtgcg ttctgcaact</span> 1741 <span class="ff_line">attaggtcaa gtgcactact tgaagtggga tgggggaaaa aaataacatc accactaaca</span> 1801 <span class="ff_line">ggggttgtat ttcactctcg tgcttgttct tcttttccta ttcttatagt ttgttggctc</span> 1861 <span class="ff_line">acctgtgcct aatcaatgtc taagtggctc gcagccgttt gtatctcccc tattcgattt</span> 1921 <span class="ff_line">ctccatcgga cggctgtagg ggctggttta tggggacctg tttcgtgcat ttttcacact</span> 1981 <span class="ff_line">attgtccata tgaaatgcac ataccacctg aagctactcc tttctcaatc tccagttgcc</span> 2041 <span class="ff_line">gctgtcttcg ttgtgtgttt gtcctttgtc agattgcaat atatatatat atatatatat</span> 2101 <span class="ff_line">gccatttatt attttaccgt gaattgtttc ttctacagat ttttgttttt tctattcttg</span> 2161 <span class="ff_line">tttatcattt cactgtgtgt gtggcgccca tcctaatcca cttggagaaa atgcagtgat</span> 2221 <span class="ff_line">atctaagtaa ctaaaaagtg agtaatgagg aagaagaaaa ttaattaaag ggcaagaggg</span> 2281 <span class="ff_line">gagggggcga aaggattgaa tgaagaaacg taagcagatg aggacatgca tctaatcgta</span> 2341 <span class="ff_line">tttgcgcaaa ggttcttggg agagggaagg aaaggagttg aaagcgtctg gtagtggtgg</span> 2401 <span class="ff_line">tggtaattac cacctgttac ggcatcagtg ctggcaccgc atgactgatt ggcgggtgac</span> 2461 <span class="ff_line">ggcccgtccc ccccttccat atttattttc gcggtgtaaa taatcaggtg ctgttttcaa</span> 2521 <span class="ff_line">aaaaaaaaaa aaaaaaaaaa aa</span> //*GC% Content for gene:
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
Resources:
See ProtocolTargetDiscoveryVDS.docx for moreEtiologic Risk Group Categories (for pathogens): http://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334
SIGMA-ALDRICH RESOURCES
Enzyme Explorer
http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer.html
Enzyme Classification Index (EC number)
http://www.sigmaaldrich.com/life-science/biochemicals/biochemical-products.html?TablePage=14573088
Databases of genes/organisms:
http://www.niaid.nih.gov/Pages/default.aspx
http://eupathdb.org/eupathdb/
https://patricbrc.vbi.vt.edu/portal/portal/patric/Home
http://www.nmpdr.org/FIG/wiki/view.cgi/Main/EssentialGenes
http://tubic.tju.edu.cn/deg/
http://csgid.org/csgid/cake/pages/community_request_gateway
http://tdrtargets.org/
http://gsc.jcvi.org/status.shtml
Scientific Nomenclature page from Center for Disease Control (gene, protein names and abbreviations)
http://wwwnc.cdc.gov/eid/pages/scientific-nomenclature.htm
Gene Information:
NCBI GENE Page: http://www.ncbi.nlm.nih.gov/gene
BLAST Page: http://blast.ncbi.nlm.nih.gov/
Protein Information:
NCBI Protein Page: http://www.ncbi.nlm.nih.gov/protein
Protein Expression Website
Protein Expression Paper: SGC_ProteinProductionPurificationNatMethods2008.pdf
Primer Overlap PCR Articles
HooverLubkowski_PCRoverlapcloninggnf042.pdf
StemmerPCRoverlapGene1995.pdf
Is my target good for Virtual Screening programs?
Reynolds_THermodynamicsLigandBinding_MedChemLett2011.pdf